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Cholangiocarcinoma (CCA) is the primary cancer of the bile duct system. The role of bile duct tissue microbiomes in CCA tumorigenesis is unestablished. To address this, sixty primary CCA tumors and matched normals, from both liver fluke (Opisthorchis viverrini) associated (OVa, n = 28) and non-O. viverrini associated (non-OVa, n = 32) cancers, were profiled using high-throughput 16S rRNA sequencing. A distinct, tissue-specific microbiome dominated by the bacterial families Dietziaceae, Pseudomonadaceae and Oxalobacteraceae was observed in bile duct tissues. Systemic perturbation of the microbiome was noted in tumor and paired normal samples (vs non-cancer normals) for several bacterial families with a significant increase in Stenotrophomonas species distinguishing tumors vs paired normals. Comparison of parasite associated (OVa) vs non-associated (non-OVa) groups identified enrichment for specific enteric bacteria (Bifidobacteriaceae, Enterobacteriaceae and Enterococcaceae). One of the enriched families, Bifidobacteriaceae, was found to be dominant in the O. viverrini microbiome, providing a mechanistic link to the parasite. Functional analysis and comparison of CCA microbiomes revealed higher potential for producing bile acids and ammonia in OVa tissues, linking the altered microbiota to carcinogenesis. These results define how the unique microbial communities resident in the bile duct, parasitic infections and the tissue microenvironment can influence each other, and contribute to cancer.

Background/aims: Global liquid chromatography mass spectrometry (LC-MS) profiling in a Thai population has previously identified a urinary metabolic signature in Opisthorchis viverrini -induced cholangiocarcinoma (CCA), primarily characterised by disturbance in acylcarnitine, bile acid, steroid, and purine metabolism. However, the detection of thousands of analytes by LC-MS in a biological sample in a single experiment potentially introduces false discovery errors. To verify these observed metabolic perturbations, a second validation dataset from the same population was profiled in a similar fashion., Methods: Reverse-phase ultra-performance liquid-chromatography mass spectrometry was utilised to acquire the global spectral profile of 98 spot urine samples (from 46 healthy volunteers and 52 CCA patients) recruited from Khon Kaen, northeast Thailand (the highest incidence of CCA globally)., Results: Metabolites were differentially expressed in the urinary profiles from CCA patients. High urinary elimination of bile acids was affected by the presence of obstructive jaundice. The urine metabolome associated with non-jaundiced CCA patients showed a distinctive pattern, similar but not identical to published studies. A panel of 10 metabolites achieved a diagnostic accuracy of 93.4% and area under the curve value of 98.8% (CI = 96.3%-100%) for the presence of CCA., Conclusions: Global characterisation of the CCA urinary metabolome identified several metabolites of biological interest in this validation study. Analyses of the diagnostic utility of the discriminant metabolites showed excellent diagnostic potential. Further larger scale studies are required to confirm these findings internationally, particularly in comparison to sporadic CCA, not associated with liver fluke infestation., (© 2022 Indian National Association for Study of the Liver.)

Background: Intrahepatic cholangiocarcinoma (iCCA) arises from bile ducts within the liver. Thailand has the highest incidence of CCA worldwide, with a high mortality rate. Early diagnosis and accurate prognostic stratification can improve overall survival. We aim to modify the AJCC/UICC 8th edition staging system for iCCA by creating the Khon Kaen University (KKU) staging system for more precise patient stratification and prognostic prediction., Methods: A total of 298 iCCA patients who underwent hepatectomy were included in this retrospective study at the Srinagarind Hospital, Khon Kaen University, Thailand. Univariate and multivariate analysis were performed to examine survival rate, hazard ratio, and prognostic factors., Results: Univariate and multivariate analysis of the cohort showed that growth patterns, histological type, histological grade, lymph node metastasis and distant metastasis were independent prognostic factors when compared to the respective reference groups. The 8th AJCC staging system incorporated growth patterns into the KKU staging system. This model modified AJCC stages I, II, and III for better prediction of patient survival., Conclusion: Growth patterns were incorporated to improve the 8th AJCC staging system for prognostication of iCCA patients in Northeast Thailand. We propose the KKU staging system as an alternative model for iCCA staging to augment the accuracy of survival prognostication., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Glycoprotein sialylation changes are associated with severe development of various cancers. We previously discovered the sialylation of serotransferrin (TF) in cholangiocarcinoma (CCA) using glycoproteomics approach. However, a simple and reliable method for validating sialylation of a specific glycobiomarker is urgently needed., Methods: We identified the altered glycosylation in CCA tissues by glycoproteomics approach using mass spectrometry. An enzyme-linked lectin assay (ELLA) was developed for determining the serum levels of sialylated TF in CCA, hepatocellular carcinoma (HCC) and healthy controls in training and validation cohorts., Results: The nine highly sialylated glycoforms of TF were markedly abundant in CCA tumor tissues than in control. Serum SNA-TF and MAL1-TF were significantly higher in CCA patients. Under receiver operating characteristic curve, serum SNA-TF concentrations significantly differentiated CCA from healthy control. Higher SNA-TF were significantly correlated with severe tumor stages and lymph node metastasis. The combined SNA-TF, MAL1-TF, and CA19-9 as a novel glycobiomarkers panel demonstrated the highest specificity (96.2%) for distinguishing CCA from HCC patients. In CCA patients with low CA19-9 levels, SNA-TF in combination with CA19-9 achieved in 97% diagnostic accuracy., Conclusions: Sialylated serotransferrin glycoforms could be used as a novel glycobiomarker for diagnosis and prediction of clinical severity in CCA patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Liver fluke infection through the consumption of raw or undercooked freshwater fish is a major public health problem in the Mekong Region. Despite the extensive efforts of liver fluke health campaigns, Northeast Thailand still reports high human infection prevalence as consumption of raw fish dishes has diminished but not ceased. This study examines the roles of social-cultural factors, particularly the influences of masculinity and misinformation, on liver fluke infection risk. Participant observation, questionnaire surveys and semi-structured interviews were conducted in four villages in Kalasin Province, Thailand, to scrutinize reasons for raw fish consumption, gender differences in raw fish culture, processes of liver fluke information dissemination, and the extent of information mismatch. Our results show that one of the key reasons (76.9%) underlying continued raw fish consumption are deeply embedded cultural practices associated with ways of rural life. About 30% of the participants indicated that they would not avoid eating raw fish, regardless of knowing the health consequences. Gender difference is evident, with 75.6% of males consuming raw fish salad (koi pla), compared to 42.7% of females. Some male participants associate raw meat consumption with virility and strength. Such beliefs underscore the cultural linkage of koi pla consumption with masculinity. Misconceptions of liver fluke life cycle and risk of infection remain, as only 15.3% of the participants correctly selected raw fish as the food source for liver fluke infection while 84.2% misunderstood that other raw foods could lead to infection. The multi-layered and hierarchical structure of public health information dissemination from medical professionals to health officers and village health volunteers to villagers has contributed to information mismatch between different layers. Our study builds on others which call for multi-pronged scientific and social strategies, as well as culturally attuned approaches to public health messaging. The study raises masculinity and misinformation as relevant considerations in disease prevention. Incorporating grounded research and gendered perspectives are part of appreciating the cultural roots of raw fish consumption. Realizing the significant role of village health volunteers in information dissemination and in supplying coherent public health messages is vital for effective health campaigns., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The colorimetric AuNPs-LISA is a new, powerful technique for the detection of Opisthorchis viverrini antigen (OvAg) in urine samples. However, the diagnostic sensitivity of the colorimetric AuNPs-LISA is powerless to screen ultralow concentrations of OvAg in urine samples in cases of early stage liver fluke infection. This work, we aimed to improve the diagnostic sensitivity of the colorimetric AuNPs-LISA by developing a new fluorescence AuNPs-LISA. O-phenylenediamine (OPD) was used as the chromogenic substrate instead of the tetramethylbenzidine (TMB) of the colorimetric AuNPs-LISA. Interestingly, the fluorescence of the OPD oxidation product by the peroxidase-like activity of labelled AuNPs can be extremely enhanced by a non-ionic surfactant, especially the Triton X-100. The proposed assay exhibited a dynamic linear detection of OvAg concentration in the range of 34.18 ng mL -1 to 273.44 ng mL -1 with the limit of detection at 36.97 ng mL -1 which the detection sensitivity enhancement around 1200-fold when comparing with the colorimetric AuNPs-LISA. This model exhibits high diagnosis sensitivity, specificity and accuracy, 91.28%, 91.75%, and 91.59%, respectively, compared to the traditional ELISA. The fluorescence AuNPs-LISA showed excellent potential for the diagnosis of OvAg in urine samples from endemic areas. This will provide an effective tool for the detection, control and elimination of human opisthorchiasis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Patients with cholangiocarcinoma (CCA) usually have no specific symptoms until an advance stage of the disease and curative treatment is not possible. Patients with early stage, operable disease can be found using ultrasonography (US). A US-screening program was implemented in Thailand where CCA incidence is the highest worldwide. Here we evaluate the effectiveness of the program by comparing the proportion of individuals with early stage CCA in the screening group with that of the walk-in group presenting at hospitals with clinical symptoms., Methods: All patients had a pathological diagnosis of CCA. The difference in the proportions and the 95% confidence interval (CI) were obtained using binomial regression., Results: Of the 762 histologically proven CCA cases, 161 were from the screening group and 601 from the walk-in group. The proportion of early stage CCA (stages 0 to II) diagnosed was 84.5% in the screening and 21.6% in the walk-in groups. After adjustment age, gender, and liver fluke infection, there was a significantly higher proportion (P < 0.001) and higher chance (P < 0.001) of having early stage CCA in the screening group than in the walk-in group., Conclusions: US-screening is an effective tool for detecting early stage, operable CCA in high incidence areas., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

The enzyme-linked immunosorbent assay (ELISA) is currently a powerful technique for the detection of Opisthorchis viverrini antigen (OvAg) in urine samples. However, its sensitivity and analysis time need to be improved. In the present study, we aimed to improve the signal enhancing system of traditional ELISA by using gold nanoparticles (AuNPs) with peroxidase-like activity on its surface instead of the horseradish peroxidase (HRP) system. The catalytic activity of the AuNPs probe can be boosted by the gold enhancing solution and the addition of ATP. The catalytic ability of the AuNPs probe depended on the probe and the H 2 O 2 concentration. The proposed approach can reduce the number of the traditional ELISA steps with better detection sensitivity. Interestingly, the limit of detection (LOD) of the test was 23.4 ng mL -1 , substantially lower than the 93.8 ng mL -1 for the traditional ELISA. The AuNPs-LISA assay showed higher sensitivity and specificity, 93.81% and 91.34%, respectively, compared to the traditional ELISA. The proposed assay was successfully applied for the detection of OvAg in urine samples. This will provide an effective tool for the detection, control and elimination of human opisthorchiasis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: A distinct serum metabonomic pattern has been previously revealed to be associated with various forms of liver disease. Here, we aimed to apply mass spectrometry to obtain serum metabolomic profiles from individuals with cholangiocarcinoma and benign hepatobiliary diseases to gain an insight into pathogenesis and search for potential early-disease biomarkers., Methods: Serum samples were profiled using a hydrophilic interaction liquid chromatography platform, coupled to a mass spectrometer. A total of 47 serum specimens from 8 cholangiocarcinoma cases, 20 healthy controls, 8 benign disease controls (bile duct strictures) and 11 patients with hepatocellular carcinoma (as malignant disease controls) were included. Data analysis was performed using univariate and multivariate statistics., Results: The serum metabolome disparities between the metabolite profiles from healthy controls and patients with hepatobiliary disease were predominantly related to changes in lipid and lipid-derived compounds (phospholipids, bile acids and steroids) and amino acid metabolites (phenylalanine). A metabolic pattern indicative of inflammatory response due to cirrhosis and cholestasis was associated with the disease groups. The abundance of phospholipid metabolites was altered in individuals with liver disease, particularly cholangiocarcinoma, but no significant difference was seen between profiles from patients with benign biliary strictures and cholangiocarcinoma., Conclusion: The serum metabolome in cholangiocarcinoma exhibited changes in metabolites related to inflammation, altered energy production and phospholipid metabolism. This study serves to highlight future avenues for biomarker research in large-scale studies., (© 2019 Indian National Association for Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Metabolic profiling, metabonomics and metabolomics are terms coined in the late 1990s as they emerged as the newest ' omics ' technology at the time. This line of research enquiry uses spectroscopic analytical platforms, which are mainly nuclear magnetic resonance spectroscopy and mass spectrometry (MS), to acquire a snapshot of metabolites, the end products of a complex biological system. Metabolic profiling enables the detection, quantification and characterisation of metabolites in biofluids, cells and tissues. The source of these compounds can be of endogenous, microbial or exogenous origin, such as dietary or xenobiotic. This results in generating extensive, multivariate spectroscopic data that require specific statistical manipulation, typically performed using chemometric and pattern recognition techniques to reduce its dimensions, facilitate its biological interpretation and allow sample classification and biomarker discovery. Consequently, it is possible to study the dynamic metabolic changes in response to disease, intervention or environmental conditions. In this review, we describe the fundamentals of MS so that clinicians can be literate in the field and are able to interrogate the right scientific questions., (© 2019 Indian National Association for Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Inflammation of the hepatobiliary system in chronic opisthorchiasis is associated with an elevated level of urinary 8-oxo-7,8 dihydro-2'deoxyguanosine (8-oxodG) during active as well as past exposure to Opisthorchis viverrini infection. In this study, we evaluated the short-term effect of praziquantel treatment on hepatobiliary disease (HBD) using urinary 8-oxodG as an inflammatory marker in a cohort of residents in endemic areas of opisthorchiasis in Khon Kaen, Thailand. The HBD status in terms of periductal fibrosis (PDF) was determined by abdominal ultrasonography and O. viverrini infection was monitored at baseline and 2-4 weeks after curative treatment by praziquantel. Analysis of O. viverrini-infected participants who were PDF-ve revealed that there was a significant reduction of urinary 8-oxodG after treatment compared with the baseline levels (p <  0.001). By contrast, in PDF+ve individuals, the levels of urinary 8-oxodG were similar between baseline and those post-treatment. Although confirmation by using a larger sample size is needed, the positive association between HBD and urinary 8-oxodG level after worm clearance suggests that chronic hepatobiliary inflammation is neither affected nor interrupted by short-term praziquantel treatment. Individuals with persistent PDF at pre- and post-treatment who have a high risk of cholangiocarcinoma, could be identified within 2-4 weeks after parasite removal by drug treatment. Thus, urinary 8-oxodG is a useful biomarker for predicting persistent PDF in individuals with a recent drug treatment history who require further clinical investigation, management and treatment., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Although cholangiocarcinoma (CCA) is usually a rare cancer, in northeast Thailand it kills 20 000 or more people every year. The prognosis is very poor owing to late stage diagnosis, with palliative treatment often representing the only option. In this area of predominantly rural Thailand, CCA is associated with infection with the liver fluke, Opisthorchis viverrini , which is classified as a group 1 carcinogen. Up to 6 million Thais are infected with this fluke. The Cholangiocarcinoma Screening and Care Program (CASCAP) was initiated in 2014 with the aim of detecting early stage CCA, allowing curative surgery, by using ultrasound (US) screening of prospectively 500 000 at risk individuals. In order to assess the massive number of radiological images, a teleconsultation system was set-up. This allows US images to be sent to a dedicated server where they can be viewed by an expert radiologist who then provides a provisional diagnosis, recommending more advanced diagnostic techniques (CT and MRI) for suspected cases. To date, 250 000 people have been screened, and 2000 cases of CCA diagnosed. This innovative information transfer procedure will also be made available to Laos, Cambodia and Vietnam, where O. viverrini infection is also common., Competing Interests: Competing interests: None declared.

Opisthorchis viverrini infection induces chronic inflammation in the bile ducts, leading to periductal fibrosis (PDF), which possibly associates to cholangiocarcinoma (CCA). Patients with CCA have a poor prognosis, which is linked to asymptomatic disease and late diagnosis. Hence, detecting early stage CCA is essential. Secretory miRNAs have been promoted as biomarkers for pathological changes associated with parasitic infections, fibrosis and/or cancer. We aimed to determine levels of miR-192 and miR-21 in the urine of O. viverrini infected, periductal fibrosis (PDF) and CCA groups using qRT-PCR. We found that miR-192 was significantly higher in O. viverrini infected, PDF and also CCA groups (p<0.05) than in healthy controls. By utilizing the Receiver Operation Characteristics (ROC) analysis, miR-192 differentiated patients with opisthorchiasis (the area under the curve; AUC=0.766), PDF subjects (AUC=0.781) and CCA patients (AUC=0.682) from healthy controls. MiR-21 was significantly higher in PDF and CCA groups (p<0.05) than in healthy controls. MiR-21 discriminated PDF subjects (AUC=0.735) and CCA patients (AUC=0.682) from healthy controls. Combined levels of these two miRNAs revealed an increased AUC of 0.812 for separating opisthorchiasis, AUC of 0.815 in discriminating PDF subjects, and AUC of 0.849 in differentiating CCA from healthy controls. Odds ratios (OR) indicated high levels of miR-192/miR-21 as risk predictors for opisthorchiasis, PDF and CCA. Levels of these miRNAs declined significantly for patients following praziquantel treatment. In conclusion, urinary miR-192/miR-21 have potential as risk indicators for opisthorchiasis and PDF-associated CCA in the endemic region., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Endothelial nitric oxide synthase (eNOS) is an isoform of the enzyme nitric oxide synthase (NOS) which is constitutively expressed in endothelial cells and plays important roles in vasodilation. We previously reported the importance of eNOS activation in cholangiocarcinoma (CCA) tissues and cell lines. The present study aims to investigate the relative abundance of eNOS and phosphorylated eNOS (P-eNOS) and their upstream regulators VEGFR3, VEGFC, EphA3 and ephrin-A1, in the Opisthorchis viverrini (Ov)/N-nitrosodimethylamine (NDMA)-induced hamster CCA model and in human CCA by semiquantitative immunohistochemical analysis of the relevant tissues. Results from the hamster model suggested an increase in eNOS and P-eNOS and upstream regulators during CCA genesis. In human CCA, high immunohistochemical staining intensity of all investigated proteins was associated with the presence of metastasis. A pairwise analysis of the staining data for eNOS and its upstream regulators showed that a concurrent increase in eNOS/VEGFR3, eNOS/ephrin-A1, eNOS/VEGFC and eNOS/EphA3 was significantly associated with metastasis. An increase in eNOS/VEGFR3, eNOS/ephrin-A1 was also associated with non-papillary type CCA. Additionally, an increase in eNOS and P-eNOS was significantly correlated with a high micro-vessel level (P=0.04). Our results indicate that the development of CCA involves upregulation of eNOS and P-eNOS and their regulators. This may drive angiogenesis and metastasis in CCA., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

L-type amino acid transporter 1 (LAT1) is highly expressed in various human cancers, including cholangiocarcinoma (CCA), the most common cancer in Northeast Thailand. Chronic inflammation and oxidative stress induced by liver fluke, Opisthorchis viverrini, infection has been recognized as the major cause of CCA in this area. We show here that an increased expression of LAT1 and its co-functional protein CD98 are found during carcinogenesis induced by Ov in hamster CCA tissues. We also demonstrate that oxidative stress induced by H 2 O 2 is time-dependent and dramatically activates LAT1 and CD98 expression in immortal cholangiocytes (MMNK1). In addition, H 2 O 2 treatment increased LAT1 and CD98 expression, as well as an activated form of AKT and mTOR in MMNK1 and CCA cell lines (KKU-M055 and KKU-M213). We also show that suppression of PI3K/AKT pathway activity with a dual PI3K/mTOR inhibitor, BEZ235, causes a reduction in LAT1 and CD98 expression in KKU-M055 and KKU-M213 in parallel with a reduction of activated AKT and mTOR. Interestingly, high expression of LAT1 in human CCA tissues is a significant prognostic factor for shorter survival. Taken together, our data show that LAT1 expression is significantly associated with CCA progression and cholangiocarcinogenesis induced by oxidative stress. Moreover, the expression of LAT1 and CD98 in CCA is possibly regulated by the PI3K/AKT signaling pathway., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The carcinogenic liver fluke Opisthorchis viverrini causes chronic inflammation in the bile duct and resulting in unremitting tissue damage that lead to hepatobiliary diseases, including cholangiocarcinoma (CCA). Despite inflammatory cytokine expression having been studied in the animal model, so far no studies have been carried out on cytokines in human CCA cases. Here we report the profile of cytokine production in peripheral blood mononuclear cells (PBMCs) collected from O. viverrini-associated human CCA and uninfected normal controls after stimulation with O. viverrini-excretory secretory (ES) product. Eleven cytokine profiles including IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNF-α and LT-α measured by flow cytometry revealed both pro-inflammatory and anti-inflammatory cytokines were increased in the O. viverrini-associated CCA compared to uninfected normal controls. Specifically, net production levels of IFN-γ, IL-10, and LT-α were 40 to >320 times higher in CCA than those of controls. These results suggest dysregulation of the immune response in the liver fluke associated CCA., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Infection with the liver fluke Opisthorchis viverrini sensu lato (s.l.), a group 1 carcinogen, is the most important risk factor for developing cholangiocarcinoma (CCA) in Southeast Asia. Cholangiocarcinoma is a fatal disease with the world's highest incidence being found in northeast Thailand. Liver fluke infection occurs through eating raw or partially cooked cyprinid fish containing metacercariae and, therefore, the control of O. viverrini s.l. infection should lead to a reduction in CCA incidence. In this report, we review and analyze the age-prevalence profile data of O. viverrini to reveal temporal changes in patterns of prevalence pre- and post-control programs in Thailand. The profiles of O. viverrini prevalence have transformed from high prevalence in school children prior to 1983 to low prevalences after 1994. This pattern strongly suggests the influence of the health education program on the likelihood of school children becoming infected. In conjunction with current developments in health and socioeconomic conditions, we predict that the incidence of CCA will be reduced with time as the population cohorts that experienced the education programs reach the age at which CCA is most likely to develop, i.e. >50 years. The lessons learned in Thailand may be applicable to other areas endemic for human liver flukes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Endogenous nitrosation due to chronic inflammation is enhanced in opisthorchiasis and plays a crucial role in the development of cholangiocarcinoma (CCA). Hepatic cytochrome P450 (CYP) family enzymes, especially CYP2A6 and CYP2E1, are involved in the metabolism of procarcinogens; these two enzymes metabolize endogenous nitrosamines to carcinogenic N-dimethylnitrosamine (NDMA). CYP2A6 activity is increased in patients infected with Opisthorchis viverrini. Our aim was to determine whether the expression and function of CYP2A6 and 2E1 in the livers of patients with O. viverrini-associated cholangiocarcinoma (CCA) was altered compared to livers without CCA. Livers of CCA patients (n = 13 cases) showed increased enzyme activities, protein and mRNA levels of CYP2A6 whereas the enzyme activity and protein levels of CYP2E1 were markedly decreased (P < 0.05). CYP2E1 mRNA levels were not altered. Large numbers of inflammatory cells and increased iNOS expression was found in areas adjacent to the tumor. The data provide evidence to support the concept that enhanced CYP2A6 activity and diminished CYP2E1 activity probably involve to the progression of CCA., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Objectives: To investigate whether Helicobacter spp. infection and the cagA of H. pylori are associated with hepatobiliary pathology, specifically biliary inflammation, cell proliferation and cholangiocarcinoma (CCA)., Methods: Helicobacter species including H. pylori, H. bilis and H. hepaticus were detected in the specimens using the polymerase chain reaction (PCR). Biliary inflammation of the liver and gallbladders was semi-quantitatively graded on hematoxylin and eosin (H&E)-stained slides. Biliary proliferation was evaluated by immunohistochemistry using the Ki-67-labelling index., Results: Helicobacter pylori was found in 66.7%, 41.5% and 25.0% of the patients in the CCA, cholelithiasis and control groups (P < 0.05), respectively. By comparison, H. bilis was found in 14.9% and 9.4% of the patients with CCA and cholelithiasis, respectively (P > 0.05), and was absent in the control group. The cagA gene of H. pylori was detected in 36.2% and 9.1% of the patients with CCA and cholelithiasis, respectively (P < 0.05). Among patients with CCA, cell inflammation and proliferation in the liver and gallbladder were significantly higher among those DNA H. pylori positive than negative., Conclusions: The present findings suggest that H. pylori, especially the cagA-positive strains, may be involved in the pathogenesis of hepatobiliary diseases, especially CCA through enhanced biliary cell inflammation and proliferation., (© 2012 International Hepato-Pancreato-Biliary Association.)